1. Field of the Invention
This invention relates to gamma-interferon and in particular to a method for purifying gamma-interferon using monoclonal and polyclonal antibodies.
2. Description of the Prior Art
Immune or gamma-interferon was originally classified on a physical basis as Type II Interferon due to its lability to acid treatment and/or heating to 56.degree. C. This operational classification distinguished it from virus-induced or Type I Interferons (alpha and beta) which, in general, are not acid or heat labile. As a result of the widespread availability of specific antisera against each of the major interferon classes (alpha, beta, and gamma), classification and distinction of each type is now usually made by serological or immunological methods. Despite this, gamma-interferon preparations are still identified as such by their rapid inactivation upon acid treatment. See, The Interferon System, 2nd edition, W. E. Stewart II, Springer-Verlag, New York, 1981.
In general, the acid inactivation process employed to identify gamma-interferon has involved reduction of the pH of a gamma-interferon containing solution to about 2, a short incubation at this pH (a few minutes to several hours), and then addition of base or dialysis against PBS to bring the pH to neutrality. Activity recoveries after such treatment are usually about 10% of control activity. To the present inventor's knowledge, there have been no reports to date showing recovery of gamma-interferon's antiviral activity to levels significantly above 10% after acid treatment.
One of the powerful tools which has been used to obtain purified solutions of selected biological materials is antibody affinity chromatography. In accordance with this technique, a chromatography column containing covalently immobilized antibodies to the biological material to be purified is prepared, and a solution containing the material, along with contaminants, is passed through the column. The immobilized antibodies form antigen-antibody complexes with the selected material which remain on the column, while the rest of the solution, including, in general, most of the contaminants, passes through the column. To free the selected biological material from the immobilized antibodies, a second solution, capable of disassociating the antigen-antibody complexes, is passed through the column. Although a variety of materials (e.g., potassium thiocyanate, urea, guanidine hydrochloride, and the like) can be included in the second solution so as to achieve the desired disassociation, the most commonly used approach is to simply make the solution acidic.
Prior to the present invention, because of gamma-interferon's known susceptibility to acid deactivation, antibody affinity chromatography has not been used to purify this important biological material. That is, although antibodies to gamma-interferon which can be immobilized on a chromatography column have been available, the art has not used affinity chromatography with gamma-interferon because there was no known way of eluting this interferon from an antibody column without destroying most of its biological activity. Acid elution, the standard elution technique, was hardly a viable approach in view of the fact that one of the basic assays for gamma-interferon involves testing for inactivation in the presence of acid. Moreover, many of the other known materials capable of producing disassociation of antigen-antibody complexes, e.g., potassium thiocyanate and urea, had also been found to inactivate gamma-interferon.
Rather than using affinity chromatography, the art turned to complicated, multi-step, purification schemes. For example, European Patent Publication No. 63,482 reports a four step purification technique run at neutral or basic pHs which includes passing a gamma-interferon containing solution through a controlled pore glass bead column, a concanavalin A-Sepharose or lentil lectin-Sepharose or pea lectin-Sepharose column, a heparin-Sepharose or procian red-Sepharose column, and a gel-filtration column. Similarly, Yip, Y. K., Barrowclough, B. S., Urban, C., and Vilcek, J., Proc. Natl. Acad. Sci. (USA), Vol. 79, pp. 1820-1824, March 1982, report a three step purification process for gamma-interferon involving a first adsorption and elution from controlled-pore glass and a second adsorption and elution from concanavalin A-Sepharose, followed by adsorptive removal of contaminating proteins on DEAE-Sephacel at pH 8.0.